胃H⁺，K⁺-ATP酶的功能表达及假定阳离子结合位点和催化中心的定点诱变

Functional expression of gastric H+,K(+)-ATPase and site-directed mutagenesis of the putative cation binding site and catalytic center.

作者信息

Asano S, Tega Y, Konishi K, Fujioka M, Takeguchi N

机构信息

Faculty of Pharmaceutical Sciences, Toyama Medical University, Japan.

出版信息

J Biol Chem. 1996 Feb 2;271(5):2740-5. doi: 10.1074/jbc.271.5.2740.

DOI:10.1074/jbc.271.5.2740

PMID:8576249

Abstract

Gastric H+,K(+)-ATPase was functionally expressed in the human kidney HEK293 cell line. The expressed enzyme catalyzed ouabain-resistant K(+)-dependent ATP hydrolysis. The K(+)-ATPase activity was inhibited by SCH 28090, a specific inhibitor of gastric proton pump, in a dose-dependent manner. By using this functional expression system in combination with site-directed mutagenesis, we investigated effects of mutations in the putative cation binding site and the catalytic center of the gastric H+,K(+)-ATPase. In Na+,K(+)-ATPase, the glutamic acid residue in the 4th transmembrane segment is regarded as one of the residues responsible for the K(+)-induced conformational change (Kuntzweiler, T. A., Wallick, E. T., Johnson, C. L., and Lingrel, J. B. (1995) J. Biol. Chem. 270, 2993-3000). When the corresponding glutamic acid (Glu-345) of H+,K(+)-ATPase was mutated to aspartic acid, lysine, or valine, the SCH 28080-sensitive K(+)-ATPase activity was abolished. However, when this residue was replaced by glutamine, about 50% of the activity was retained. This mutant showed a 10-fold lower affinity for K+ (Km = 2.6 mM) compared with the wild-type enzyme (Km = 0.24 mm). Thus, Glu-345 is important in determining the K+ affinity of H+,K(+)-ATPase. When the aspartic acid residue in the phosphorylation site was mutated to glutamic acid, this mutant showed no SCH 28080-sensitive K(+)-ATPase activity. Thus, amino acid replacement of the phosphorylation site is not tolerated and a stringent structure appears to be required for enzyme activity. When the lysine residue in the fluorescein isothiocyanate binding site (part of ATP binding site) was mutated to arginine, asparagine, or glutamic acid, the SCH 28080-sensitive K(+)-ATPase activity was eliminated. However, the mutant in which this residue was changed to glutamine had about 30% of the activity, suggesting that amino acid replacement of this site is tolerated to a certain extent.

摘要

胃H⁺，K⁺-ATP酶在人肾HEK293细胞系中实现了功能表达。所表达的酶催化了哇巴因抗性的K⁺依赖性ATP水解。K⁺-ATP酶活性受到胃质子泵特异性抑制剂SCH 28090的剂量依赖性抑制。通过将这种功能表达系统与定点诱变相结合，我们研究了胃H⁺，K⁺-ATP酶推定的阳离子结合位点和催化中心突变的影响。在Na⁺，K⁺-ATP酶中，第4个跨膜段中的谷氨酸残基被认为是负责K⁺诱导的构象变化的残基之一（Kuntzweiler，T.A.，Wallick，E.T.，Johnson，C.L.，和Lingrel，J.B.（1995）J.生物化学.270，2993 - 3000）。当H⁺，K⁺-ATP酶相应的谷氨酸（Glu - 345）突变为天冬氨酸、赖氨酸或缬氨酸时，对SCH 28080敏感的K⁺-ATP酶活性被消除。然而，当该残基被谷氨酰胺取代时，约50%的活性得以保留。与野生型酶（Km = 0.24 mM）相比，该突变体对K⁺的亲和力低10倍（Km = 2.6 mM）。因此，Glu - 345在决定H⁺，K⁺-ATP酶的K⁺亲和力方面很重要。当磷酸化位点的天冬氨酸残基突变为谷氨酸时，该突变体没有表现出对SCH 28080敏感的K⁺-ATP酶活性。因此，磷酸化位点的氨基酸替换是不被允许的，酶活性似乎需要严格的结构。当异硫氰酸荧光素结合位点（ATP结合位点的一部分）中的赖氨酸残基突变为精氨酸、天冬酰胺或谷氨酸时，对SCH 28080敏感的K⁺-ATP酶活性被消除。然而，该残基变为谷氨酰胺的突变体具有约30%的活性，这表明该位点的氨基酸替换在一定程度上是被允许的。